T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes

Abstract Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Blood ◽

10.1182/blood.v126.23.420.420 ◽

2015 ◽

Vol 126 (23) ◽

pp. 420-420

Author(s):

Melissa M. Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley Elizabeth Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Severe Thrombocytopenia ◽

Extrinsic Factors ◽

Platelet Production ◽

Platelet Counts

Abstract Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Bone Marrow Macrophage Galectin-3 Regulates Platelet Production through Recognition of O-Glycans on Megakaryocytes

Blood ◽

10.1182/blood.v128.22.409.409 ◽

2016 ◽

Vol 128 (22) ◽

pp. 409-409

Author(s):

Melissa M Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley E Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Antigen Expression ◽

Blood Platelet ◽

Activated Macrophages ◽

Bone Marrow Macrophage ◽

Platelet Production ◽

Platelet Counts ◽

Pathological Conditions ◽

Galectin 3

Abstract Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies. The TF antigen is a disaccharide presented in cryptic form on O-glycans and covered by a sialic acid moiety. The sialyltransferase ST3Gal1 transfers sialic acid onto the TF antigen. To investigate the role of O-glycans in thrombopoiesis, we generated mice with increased TF antigen in MKs by generating St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the MK lineage. As expected, St3gal1loxP/PF4+ circulating platelets and BM MKs had increased TF antigen expression, compared to controls, as evidenced by peanut agglutinin (PNA) binding. Other blood cell lineages had no increase in TF antigen expression. St3gal1loxP/PF4+ mice developed mild thrombocytopenia, but surprisingly had virtually normal platelet clearance. BM MK colony forming units and in vitro proplatelet production were normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+BM environment affected platelet production. St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, indicative of an increase in phagocytic macrophages. In vivo macrophage ablation by injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages, thereby normalizing blood platelet counts and size. Flow cytometric phenotypic analysis of BM-derived macrophages showed an increased population of activated macrophages in St3gal1loxP/PF4+ mice, compared to controls, specifically macrophages with increased galectin-3 expression, a ligand for the TF antigen. Immunofluorescence staining of BM sections using a specific antibody towards the TF antigen showed that MK progenitors and pro-platelet-like structures expressed TF antigen in control BMs, which is significantly increased in St3gal1loxP/PF4+ mice and co-localized with galectin-3 expressing macrophages, supporting the notion that MK O-glycans and macrophage galectin-3 play a role in thrombopoiesis under steady state and pathological conditions. Consistent with this notion, galectin-3 deficient mice have slightly, but significantly increased blood platelet counts. We conclude that galactin-3 plays a minor role in normal thrombopoiesis. Activation of galectin-3 expressing macrophages by the MK TF antigen leads to MK phagocytosis, inhibition of platelet formation and thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice

Blood ◽

10.1182/blood.v81.4.901.901 ◽

1993 ◽

Vol 81 (4) ◽

pp. 901-908 ◽

Cited By ~ 115

Author(s):

TY Neben ◽

J Loebelenz ◽

L Hayes ◽

K McCarthy ◽

J Stoudemire ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Color Flow ◽

Platelet Counts ◽

In Vivo Treatment ◽

Interleukin 11 ◽

Megakaryocyte Progenitors

Abstract The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.

Download Full-text

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Blood ◽

10.1182/blood.v114.22.3622.3622 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3622-3622

Author(s):

Frederick Karl Racke ◽

Maureen E Baird ◽

Rolf Barth ◽

Tianyao Huo ◽

Weilian Yang ◽

...

Keyword(s):

Conflicts Of Interest ◽

Leukemic Cell ◽

The Novel ◽

Drug Induced ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Counts ◽

Pkc Isoforms

Abstract Abstract 3622 Poster Board III-558 Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Thrombocytopenia In Severe Anemia of Iron Deficiency

Blood ◽

10.1182/blood.v116.21.5153.5153 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5153-5153

Author(s):

Getinet D. Ayalew ◽

Juhi Mittal ◽

Ratesh Khillan ◽

Miriam Kim ◽

Albert S. Braverman ◽

...

Keyword(s):

Bone Marrow ◽

Iron Deficiency ◽

African American Women ◽

Conflicts Of Interest ◽

Ferrous Sulphate ◽

Bone Marrow Aspiration ◽

Deficiency Anemia ◽

Platelet Counts ◽

Cell Counts ◽

Erythroid Hyperplasia

Abstract Abstract 5153 Introduction: Iron deficiency suppresses hemoglobin synthesis and erythropoiesis, but the resulting anemia is frequently associated with thrombocytosis. Methods: The clinical and hematologic data of seven women with severe iron deficiency anemia (IDA) and thrombocytopenia were retrospectively analyzed. Results: All patients were African-American women with symptomatic IDA, due to bleeding from uterine fibroids in 6 and from colonic diverticulosis in 1. They were 31–70 years of age, median 38. None had palpable splenomegaly. Hemoglobin ranged from 2.9–5.5, median 4.2 g/dL. MCV ranged from 57–70 fl, median 68. Absolute reticulocyte counts ranged from 19,000 – 23, 000/mm3. The initial serum ferritin ranged from 2 to 42 ng/ml, median 4. Serum iron levels ranged from 10 to 70 mcg/dl with median 30, while iron-binding capacities ranged from 381–426 mcg/dl. Serum erythropoietin (EPO) levels were >2000U/ml in two of the patients. Serum lactic dehydrogenase, bilirubin levels and liver function tests were normal; and Coombs' test negative in all cases. White blood cell counts were normal. The platelet counts ranged from 12 to 103, with a median of 46 × 109/L. Peripheral blood smears showed microcytic hypochromic red blood cells (RBC), with no evidence of platelet clumping. Bone marrow aspiration and biopsy on two patients showed increased numbers of normal megakaryocytes, erythroid hyperplasia and absent iron stores. Six patients were treated with packed RBC transfusions, and ferrous sulphate 325 mg orally was initiated at presentation in 7. Their thrombocytopenia was not treated with steroids or other agents. Three patients' platelet count reached normal or super-normal levels within 72 hours. Six patients were seen at ≥3 months after presentation, and all had achieved normal platelet counts and hemoglobin. Conclusions: These data imply that severe IDA can sometimes cause thrombocytopenia rather than thromobocytosis. We cannot be sure whether these patients' uniform normalization of platelet counts was due to treatment of their anemia by transfusion, or iron therapy. Though bone marrow megakaryocyte numbers were increased in 2 patients, there is no evidence for peripheral platelet destruction. Platelet release from megakaryocytes may have decreased in these patients. Pharmacologic EPO therapy can occasionally cause thrombocytopenia, and high endogenous EPO levels in our patients may have reduce their platelet counts. This conclusion is consistent with their apparent response to transfusion. Though the pathogenesis of IDA-associated thrombocytopenia is not known, our data suggest that the results of anemia and iron deficiency treatment should be evaluated before investigating thrombocytopenia as an independent problem. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Endothelial Cell Secretion Combined with Shear Stress Strongly Increase Platelet Production by Human Megakaryocytes,

Blood ◽

10.1182/blood.v118.21.3411.3411 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3411-3411

Author(s):

Yasmine Ouzegdouh ◽

Laurence Momeux ◽

Claude Capron ◽

Elisabeth Cramer Bordé

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Shear Stress ◽

Conflicts Of Interest ◽

Video Microscopy ◽

Platelet Production ◽

Platelet Size ◽

Platelet Release

Abstract Abstract 3411 It is well established that megakaryocytes (MK), in order to achieve terminal maturation in the bone marrow, migrate from the osteoblastic niche to the vascular niche, close to medullary sinusoids. It is also admitted that, in order to release platelets, MK extend cytoplasmic extensions in the lumen of the sinusoid followed by platelet release. Therefore, we have investigated the role of endothelial cells combined with shear stress on MK late differentiation steps and platelet production. To do so, human MK were grown from umbilical cord blood or bone marrow CD34+ cells, in the presence of Stem Cell Factor (SCF) and thrombopoietin for 10 days. Then they were co-cultured with human endothelial cells (HUVEC) for 3 days and analysed by video-microscopy, flow cytometry, immunofluorescence and electron microscopy. In these conditions, most MK (>50%) extended numerous and prominent proplatelets: immunofluorescence showed virtually complete unwinding of their cytoplasm, which extended in the form of long proplatelets; this rarely occurred in control MK. Electron microscopy showed that these MK were formed by a coarse chromatin nucleus surrounded by a thin cytoplasmic ring and surrounded by platelet-size territories displaying alpha-granules, mitochondria and canalicular system which coincided to the sections of the proplatelet swellings and to the unwinding of the demarcation membrane system. Another type of experiment of co-culture MK/endothelial cells was conducted in transwells and led to similar results, ie, increase of MK cytoplasmic maturation and proplatelet formation indicating that cell/cell contact was not necessary for up-regulating proplatelet production, rather that (a) soluble product(s) was(ere) secreted from endothelial cells. Flow cytometry, at this step, failed to demonstrate significant changes in the platelet production rate. We then submitted MK co-cultured with endothelial cells to shear stress and examined them by flow cytometry and video microscopy. We could then demonstrate that platelet release was strongly increased (× 3.8 ± 0.9, n=3) when MK had been in contact with endothelial cells compared to control MK. The released platelet-size particles expressed CD41 and, when stimulated by thrombin, were also able to express CD62P. Video-microscopy confirmed that proplatelet and platelet shedding occurred after exposition of mature MK to shear stress. When examined by video-microscopy, live MK whose DNA had been stained with the fluorescent dye Hoechst 33342 showed that nuclear lobes separated under high shear stress: indeed they became located at opposite poles of the cell, while the cytoplasmic volume extended and elongated, becoming organized in proplatelets which exhibited a succession of platelet size subunits; eventually cytoplasmic scission occurred in parallel with MK nuclear lobe segregation in distinct cell fractions, each carrying proplatelets; proplatelets subsequently fractionated and were then released from the cell core containing the fluorescent nuclear lobe. Thus shear stress leads both to cytoplasm and nucleus fragmentation. This is a dynamic explanation to the fact that entire MK nuclei are rarely found in the human bone marrow. This observation also gives sense to the unique phenomenon of MK polyploidy. In conclusion, this study indicates that endothelial microenvironment combined with circulatory shear forces are determinant up-regulating factors which increase platelet production. It also shows that shear stress is able to induce nuclear as well as cytoplasmic MK fragmentation, leading to a new anatomical concept of circulating platelet shedding MK subunits. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation

Blood ◽

10.1182/blood.v120.21.853.853 ◽

2012 ◽

Vol 120 (21) ◽

pp. 853-853

Author(s):

Satya P. Kunapuli ◽

John C Kostyak

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Dna Content ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Platelet Counts ◽

Cell Counts ◽

Apoptotic Protein ◽

Primary Mouse ◽

Marrow Cells

Abstract Abstract 853 Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Platelet Biogenesis in the Lung Circulation

Blood ◽

10.1182/blood-2018-99-109544 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-22-SCI-22

Author(s):

Mark Looney

Keyword(s):

Bone Marrow ◽

Lung Transplant ◽

Conflicts Of Interest ◽

Direct Proof ◽

Mouse Lung ◽

Mouse Strains ◽

Hematopoietic Progenitors ◽

Platelet Production ◽

Platelet Counts ◽

Transplant Procedure

Abstract Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals (c-mpl-/-) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl-/-animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Bortezomib Induced Thrombocytopenia Might Be Due to the Inhibition of Proplatelet Formation of Megakaryocyte.

Blood ◽

10.1182/blood.v116.21.3696.3696 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3696-3696

Author(s):

Kazunori Murai ◽

Shugo Kowata ◽

Akiko Abo ◽

Tatsuo Oyake ◽

Kenichi Nomura ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Studies ◽

Bone Marrow Cells ◽

Platelet Production ◽

Platelet Counts ◽

Proplatelet Formation ◽

Marrow Cells

Abstract Abstract 3696 Background: Bortezomib is potent and reversible proteasome inhibitor that has been extensively used for multiple myeloma. Several clinical studies demonstrated that overall response rates using bortezomib alone to relapsed or refractory patients with multiple myeloma were 33 to 50%. The most common grade 3 adverse event was a cyclic thrombocytopenia, which was reported in 20–30% of patients in several clinical studies. The mechanism by which bortezomib causes thrombocytopenia remains unknown. In this study, we evaluated the effect of bortezomib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method: All animal procedures were approved by the Institutional Animal Care and Use Committee in Iwate Medical University. Male ddY at 8 weeks of age mice were used in all experiments. In vivo experiments: (a) The mice received 2.5 mg/kg bortezomib via tail-vein injection. Blood was obtained and the following experiments were carried out at day 2, 4, 6, 8, 10 after intravenous injection (n=9, each group). Complete blood counts were measured. Reticulated platelet (RP) was analyzed by flow cytometry using thiazole orange (TO) to evaluate platelet kinetics. Plasma TPO level were measured by ELISA. Bone marrow megakaryocyte's number and morphology from femur in bortezomib- and control-treated mice were observed by microscopy. Femur was fixed in 10% buffered formalin, decalcificated, embedded in paraffin and stained for Hematoxylin-Eosin (H-E). (b) Bortezomib (2.5 mg/kg) was administrated via tail-vein to mice. After 24hr, bone marrow cells were cultured in MegaCult®-C at 5% CO2 and 20% O2for 7 days. The megakaryocytic colonies (CFU-Megs-in vivo) were counted. In vitro experiments: (c) Bone marrow cells, obtained from non-treated mice, were cultured at 37°C in 5% CO2and 20% O2 for 7 days with bortezomib (0.01, 0.1, 1, 10, 100 ng/ml). CFU-Megs were counted (CFU-Megs-in vitro). (d) Proplatelet formation: Murine megakarocytes were partially purified from bone marrow using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte)well) and cultured in IMDM in duplicates, supplemented with 1 × ITS-G (Life technologies) and each concentration of bortezomib (0.01, 0.1, 1, 10, 100 ng/ml), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Results: (a) Control mice did not have any significant change in platelet counts, % reticulated platelets and plasma TPO levels at days 0, 2, 4, 6, 8, 10. While, bortezomib treated mice (2.5mg/kg) had a significant reduction in platelet counts at day 2 (470 ± 210 × 109/L. P<0.001), at day 4 (667 ± 118 × 109/L, P<0.001). The platelet counts returned to normal value at day 6 (903 ± 548 × 109/L) and day 10 (1122 ± 187 × 109/L). RP (%) began to increase at day 6 (8.8 ± 4.0 %). Plasma TPO levels tend to increase at day 4. Means megakaryocytes's number in one field of femur was similar in between bortezomib non-treated and –treated mice. The megakaryocytes were similar in morphology at each day, too. (b) CFU-Megs-in vivo were similar in number between bortezomib non-treated and –treated mice (38.0 ± 6.1 vs 34.5 ± 5.6 per 1 × 105 bone marrow cells respectively). (c) CFU-Megs-in vitro were not decreased significantly at 0.001 to 1 ng/ml and decreased significantly (p<0.01) at 10 and 100 ng/ml of bortezomib. (d) Proplatelet formation (PPF) were decreased significantly at 0.01, 0.1, 1, 10, 100 ng/ml bortezomib (0 mg)ml: 25.2 ± 4.8%, 0.01ng/ml: 23.8 ± 4.9%, 0.1 ng/ml: 18.4 ± 3.1% p<0.01, 1 ng/ml: 13.2 ± 3.8% p<0.001, 10 ng/ml: 13.3 ± 2.1% p<0.001, 100ng/ml: 5.9 ± 1.4 % p<0.001). Discussion & Conclusion: Bortezomib did not adversely affect on megakaryocytic prognitors nor megakaryocytes. It did inhibit PPF, that is, the step of platelet production, even when bortezomib plasma concentration levels have gone down. Plasma TPO level showed an inverse relationship against circulating platelet counts. Based on the evidence in which Cmax of plasma bortezomib concentration was under 100 ng/ml in bortezomib-injected mice (2.5mg/kg), bortezomib induced thrombocytopenia might be due to the inhibition of proplatelet formation of megakaryocyte. Disclosures: No relevant conflicts of interest to declare.

Download Full-text